Cyclosporine A and IFNγ licencing enhances human mesenchymal stromal cell potency in a humanised mouse model of acute graft versus host disease.

Immunosuppressive ability in human MSC donors has been shown to be variable and may be a limiting factor in MSC therapeutic efficacy in vivo. The importance of cytokine activation of mesenchymal stromal cells (MSCs) to facilitate their immunosuppressive function is well established. This study sought to further understand the interactions between MSCs and the commonly used calcineurin inhibitor cyclosporine A (CsA). The existing literature regarding approaches that use MSCs and cyclosporine are conflicting regarding the effect of CsA on MSC potency and function. Here, we clearly demonstrate that when added at the same time as MSCs, CsA negatively affects MSC suppression of T cell proliferation. However, licencing MSCs with IFNγ before addition of CsA protects MSCs from this negative effect. Notably, adding CsA to MSCs after IFNγ pre-stimulation enhances MSC production of IDO. Mechanistically, we identified that CsA reduces SOCS1 expression to facilitate enhanced IDO production in IFNγ pre-stimulated MSCs. Importantly, CsA exposure to IFNγ pre-stimulated MSC before administration, significantly enhanced the potency of MSCs in a human relevant humanised mouse model of acute Graft versus Host Disease. In summary, this study identified a novel licencing strategy to enhance MSC potency in vitro and in vivo.

Drug delivery formulation impacts cyclosporine efficacy in a humanised mouse model of acute graft versus host disease.

Acute graft versus host disease (aGvHD) is an allogeneic T cell mediated disease which manifests as a severe inflammatory disease affecting multiple organs including the liver, skin, lungs and gastrointestinal tract. Existing prophylactic and therapeutic approaches in aGvHD include the use of cyclosporine A (CyA), however the currently approved CyA formulations which were designed to optimise systemic CyA bioavailability can have a number of side effects including nephrotoxicity as well as the potential to attenuate the beneficial Graft-versus-Leukemia (GvL) effect. An added complication with CyA is that it has a narrow therapeutic window, and following oral administration is absorbed only from the small intestine, with variable cytochrome P450 metabolism contributing to intra- and inter-patient variability. This study sought to investigate the efficacy of a novel CyA oral formulation enabled by the integrated SmPill® oral drug delivery platform in a humanised mouse model of aGvHD. The study compared the approved optimised CyA (Neoral®) with SmPill®-enabled CyA and a systemic intravenous CyA formulation. Our findings clearly demonstrate superior efficacy of the novel SmPill® CyA in prolonging survival in a clinically relevant humanised aGvHD model. SmPill® CyA significantly reduced pathological score in the small intestine, colon, liver and lung of aGvHD mice. In addition, SmPill® CyA significantly reduced the levels of pro-inflammatory cytokines in all the GvHD target tissues examined. Notably, SmPill® CyA was significantly more potent in reducing GvHD associated pathology and inflammatory cytokine production compared to the optimised approved oral CyA formulation, Neoral®.

Encapsulated cyclosporine does not change the composition of the human microbiota when assessed ex vivo and in vivo.

Introduction. Management of steroid-refractory ulcerative colitis has predominantly involved treatment with systemic cyclosporine A (CyA) and infliximab.Aim. The purpose of this study was to assess the effect of using a colon-targeted delivery system CyA formulation on the composition and functionality of the gut microbiota.Methodology. Ex vivo faecal fermentations from six healthy control subjects were treated with coated minispheres (SmPill) with (+) or without (-) CyA and compared with a non-treated control in a model colon system. In addition, the in vivo effect of the SmPill+CyA formulation was investigated by analysing the gut microbiota in faecal samples collected before the administration of SmPill+CyA and after 7 consecutive days of administration from eight healthy subjects who participated in a pilot study.Results. Analysis of faecal samples by 16S rRNA gene sequencing indicated little variation in the diversity or relative abundance of the microbiota composition before or after treatment with SmPill minispheres with or without CyA ex vivo or with CyA in vivo. Short-chain fatty acid profiles were evaluated using gas chromatography, showing an increase in the concentration of n-butyrate (P=0.02) and acetate (P=0.32) in the faecal fermented samples incubated in the presence of SmPill minispheres with or without CyA. This indicated that increased acetate and butyrate production was attributed to a component of the coated minispheres rather than an effect of CyA on the microbiota. Butyrate and acetate levels also increased significantly (P=0.05 for both) in the faecal samples of healthy individuals following 7 days' treatment with SmPill+CyA in the pilot study.Conclusion. SmPill minispheres with or without CyA at the clinically relevant doses tested here have negligible direct effects on the gut microbiota composition. Butyrate and acetate production increased, however, in the presence of the beads in an ex vivo model system as well as in vivo in healthy subjects. Importantly, this study also demonstrates the relevance and value of using ex vivo colon models to predict the in vivo impact of colon-targeted drugs directly on the gut microbiota.

Type I IFN signalling is required for cationic adjuvant formulation (CAF)01-induced cellular immunity and mucosal priming.

Despite being in the midst of a global pandemic of infections caused by the pathogen Chlamydia trachomatis, a vaccine capable of inducing protective immunity remains elusive. Given the C. trachomatis mucosal port of entry, a formulation compatible with mucosal administration and capable of eliciting potent genital tract immunity is highly desirable. While subunit vaccines are considered safer and better tolerated, these are typically poorly immunogenic and require co-formulation with immune-potentiating adjuvants. However, of the adjuvants licensed for use in humans, very few drive robust cellular responses, a pre-requisite for protection against C. trachomatis infection. Recently, the cationic adjuvant formulations (CAF) have been shown to induce robust humoral and cellular immunity in pre-clinical models of chlamydia, malaria and tuberculosis (TB). Here, we demonstrate that CAF01 induces potent immune responses when combined with the major outer membrane protein (MOMP) of C. trachomatis following parenteral immunisation and also as part of a heterologous prime/boost regime. We show that a subcutaneous prime with CAF01-adjuvanted recombinant MOMP licenses antigen-specific immunity at distant mucosal sites which can be activated following oral antigen re-encounter in the absence of concomitant adjuvant stimulation. Finally, we shed light on the mechanism(s) through which CAF01 elicits robust antigen-specific immunity to co-formulated MOMP via type I interferon (IFN) signalling.

An oral alpha-galactosylceramide adjuvanted Helicobacter pylori vaccine induces protective IL-1R- and IL-17R-dependent Th1 responses.

Helicobacter pylori causes chronic gastric infection that can lead to peptic ulcers and is an identified risk factor for gastric cancer development. Although much effort has been put into the development of a Helicobacter pylori vaccine over the last three decades, none has yet reached clinical application. Specific challenges pertaining to effective H. pylori vaccine development include the lack of proven vaccine-effective antigens and safe mucosal adjuvants to enhance local immune responses as well as the lack of accepted correlates of protection. Herein, we demonstrate that prophylactic intragastric immunisation with a whole-cell killed H. pylori antigen administered together with the non-toxic oral adjuvant α-galactosylceramide (α-GalCer) induced effective immune protection against H. pylori infection in mice, which was of similar magnitude as when using the "gold standard" cholera toxin as adjuvant. We further describe that this α-GalCer-adjuvanted vaccine formulation elicited strong intestinal and systemic Th1 responses as well as significant antigen-specific mucosal and systemic antibody responses. Finally, we report that the protective intestinal Th1 responses induced by α-GalCer are dependent on CD1d, IL-1R as well as IL-17R signalling. In summary, our results show that α-GalCer is a promising adjuvant for inclusion in an oral vaccine against H. pylori infection.

Hypoxia-inducible factor hydroxylase inhibition enhances the protective effects of cyclosporine in colitis.

Inflammatory bowel disease (IBD) is characterized by epithelial barrier dysfunction with resultant inflammation as the mucosal immune system becomes exposed to luminal antigens. The hydroxylase inhibitor dimethyloxalylglycine (DMOG) reduces symptoms in experimental colitis through the upregulation of genes promoting barrier function and inhibition of epithelial cell apoptosis. The immunosuppressive drug cyclosporine reduces inflammation associated with IBD via suppression of immune cell activation. Given the distinct barrier protective effect of DMOG and the anti-inflammatory properties of cyclosporine, we hypothesized that combining these drugs may provide an enhanced protective effect by targeting both barrier dysfunction and inflammation simultaneously. We used the dextran sulfate sodium model of colitis in C57BL/6 mice to determine the combinatorial efficacy of cyclosporine and DMOG. While cyclosporine and DMOG ameliorated disease progression, in combination they had an additive protective effect that surpassed the level of protection afforded by either drug alone. The ability of DMOG to augment the anti-inflammatory effects of cyclosporine was largely due to preservation of barrier function and at least in part due to zonula occludens-1 regulation. We propose that combining the barrier protective effects of a hydroxylase inhibitor with the anti-inflammatory effects of cyclosporine provides added therapeutic benefit in colitis.NEW & NOTEWORTHY Inflammatory bowel disease is the result of decreased intestinal epithelial barrier function leading to exposure of the mucosal immune system to luminal antigens causing inflammation, which in turn further decreases epithelial barrier function. We demonstrate for the first time that strengthening the epithelial barrier with a hydroxylase inhibitor in combination with the administration of the immunosuppressive cyclosporine provides additive therapeutic advantage in a murine model of colitis.

Alpha-galactosylceramide enhances mucosal immunity to oral whole-cell cholera vaccines.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae) that results in 3-4 million cases globally with 100,000-150,000 deaths reported annually. Mostly confined to developing nations, current strategies to control the spread of cholera include the provision of safe drinking water and improved sanitation and hygiene, ideally in conjunction with oral vaccination. However, difficulties associated with the costs and logistics of these strategies have hampered their widespread implementation. Specific challenges pertaining to oral cholera vaccines (OCVs) include a lack of safe and effective adjuvants to further enhance gut immune responses, the complex and costly multicomponent vaccine manufacturing, limitations of conventional liquid formulation and the lack of an integrated delivery platform. Herein we describe the use of the orally active adjuvant α-Galactosylceramide (α-GalCer) to strongly enhance intestinal bacterium- and toxin-specific IgA responses to the OCV, Dukoral® in C57BL/6 and BALB/c mice. We further demonstrate the mucosal immunogenicity of a novel multi-antigen, single-component whole-cell killed V. cholerae strain and the enhancement of its immunogenicity by adding α-GalCer. Finally, we report that combining these components and recombinant cholera toxin B subunit in the SmPill® minisphere delivery system induced strong intestinal and systemic antigen-specific antibody responses.

Thermostability of the coating, antigen and immunostimulator in an adjuvanted oral capsule vaccine formulation.

Oral vaccines present an attractive alternative to injectable vaccines for enteric diseases due to ease of delivery and the induction of intestinal immunity at the site of infection. However, susceptibility to gastrointestinal proteolysis, limited transepithelial uptake and a lack of clinically acceptable adjuvants present significant challenges. A further challenge to mass vaccination in developing countries is the very expensive requirement to maintain the cold chain. We recently described the effectiveness of a Single Multiple Pill® (SmPill®) adjuvanted capsule approach to enhance the effectiveness of a candidate enterotoxigenic Escherichia coli (ETEC) oral vaccine. Here it was demonstrated that this delivery system maintains the antigenicity of ETEC colonisation factor antigen I (CFA/I) and the immunostimulatory activity of the orally active α-Galactosylceramide (α-GalCer) adjuvant after storage of SmPill® minispheres under room temperature and extreme storage conditions for several months. In addition, the internal structure of the cores of SmPill® minispheres and antigen release features at intestinal pH were found to be preserved under all these conditions. However, changes in the surface morphology of SmPill® minispheres leading to the antigen release at gastric pH were observed after a few weeks of storage under extreme conditions. Those modifications were prevented by the introduction of an Opadry® White film coating layer between the core of SmPill® minispheres and the enteric coating. Under these conditions, protection against antigen release at gastric pH was maintained even under high temperature and humidity conditions. These results support the potential of the SmPill® minisphere approach to maintain the stability of an adjuvanted whole cell killed oral vaccine formulation.

Coated minispheres of salmon calcitonin target rat intestinal regions to achieve systemic bioavailability: Comparison between intestinal instillation and oral gavage.

Achieving oral peptide delivery is an elusive challenge. Emulsion-based minispheres of salmon calcitonin (sCT) were synthesized using single multiple pill (SmPill®) technology incorporating the permeation enhancers (PEs): sodium taurodeoxycholate (NaTDC), sodium caprate (C10), or coco-glucoside (CG), or the pH acidifier, citric acid (CA). Minispheres were coated with an outer layer of Eudragit® L30 D-55 (designed for jejunal release) or Surelease®/Pectin (designed for colonic release). The process was mild and in vitro biological activity of sCT was retained upon release from minispheres stored up to 4months. In vitro release profiles suggested that sCT was released from minispheres by diffusion through coatings due to swelling of gelatin and the polymeric matrix upon contact with PBS at pH6.8. X-ray analysis confirmed that coated minispheres dissolved at the intended intestinal region of rats following oral gavage. Uncoated minispheres at a dose of ~2000I.U.sCT/kg were administered to rats by intra-jejunal (i.j.) or intra-colonic (i.c.) instillation and caused hypocalcaemia. Notable sCT absolute bioavailability (F) values were: 5.5% from minispheres containing NaTDC (i.j), 17.3% with CG (i.c.) and 18.2% with C10 (i.c.). Coated minispheres administered by oral gavage at threefold higher doses also induced hypocalcaemia. A highly competitive F value of 2.7% was obtained for orally-administered sCT-minispheres containing CG (45µmol/kg) and coated with Eudragit®. In conclusion, the SmPill® technology is a potential dosage form for several peptides when formulated with PEs and coated for regional delivery. PK data from instillations over-estimates oral bioavailability and poorly predicts rank ordering of formulations.

A novel adjuvanted capsule based strategy for oral vaccination against infectious diarrhoeal pathogens.

Diarrhoeal infections are a major cause of morbidity and mortality with enterotoxigenic Escherichia coli (ETEC) and cholera imposing a significant global burden. There is currently no licensed vaccine for ETEC. Development of new nonliving oral vaccines has proven difficult due to the physicochemical and immunological challenges associated with the oral route. This demands innovative delivery solutions to protect antigens, control their release and build in immune-stimulatory activity. We describe the Single Multiple Pill® (SmPill®) vaccine formulation which combines the benefits of enteric polymer coating to protect against low gastric pH, a dispersed phase to control release and aid the solubility of non-polar components and an optimized combination of adjuvant and antigen to promote mucosal immunity. We demonstrate the effectiveness of this system with whole cell killed E. coli overexpressing colonization factor antigen I (CFA/I), JT-49. Alpha-galactosylceramide was identified as a potent adjuvant within SmPill® that enhanced the immunogenicity of JT-49. The bacteria associated with the dispersed phase were retained within the capsules at gastric pH but released at intestinal pH. Vaccination with an optimized SmPill® formulation promoted CFA/I-specific immunoglobulin A (IgA) responses in the intestinal mucosa in addition to serum IgG and a solubilized adjuvant was indispensable for efficacy.

Enhanced colonic delivery of ciclosporin A self-emulsifying drug delivery system encapsulated in coated minispheres.

OBJECTIVES: Investigate the potential of coated minispheres (SmPill®) to enhance localized Ciclosporin A (CsA) delivery to the colon. METHODS: CsA self-emulsifying drug delivery systems (SEDDS) were encapsulated into SmPill® minispheres. Varying degrees of coating thickness (low, medium and high) were applied using ethylcellulose and pectin (E:P) polymers. In vitro CsA release was evaluated in simulated gastric and intestinal media. Bioavailability of CsA in vivo following oral administration to pigs of SmPill® minispheres was compared to Neoral® po and Sandimmun® iv in a pig model. CsA concentrations in blood and intestinal tissue were determined by HPLC-UV. RESULTS: In vitro CsA release from coated minispheres decreased with increasing coating thickness. A linear relationship was observed between in vitro CsA release and in vivo bioavailability (r(2) = 0.98). CsA concentrations in the proximal, transverse and distal colon were significantly higher following administration of SmPill®, compared to Neoral® po and Sandimmun® iv (p < 0.05). Analysis of transverse colon tissue subsections also revealed significantly higher CsA concentrations in the mucosa and submucosa using SmPill® minispheres (p < 0.05). CONCLUSIONS: Modulating E:P coating thickness controls release of CsA from SmPill® minispheres. Coated minispheres limited CsA release in the small intestine and enhanced delivery and uptake in the colon. These findings demonstrate clinical advantages of an oral coated minisphere-enabled CsA formulation in the treatment of inflammatory conditions of the large intestine.

In vitro and in vivo preclinical evaluation of a minisphere emulsion-based formulation (SmPill®) of salmon calcitonin.

Salmon calcitonin (sCT, MW 3432Da) is a benchmark molecule for an oral peptide delivery system because it is degraded and has low intestinal epithelial permeability. Four dry emulsion minisphere prototypes (SmPill®) containing sCT were co-formulated with permeation enhancers (PEs): sodium taurodeoxycholate (NaTDC), sodium caprate (C10) or coco-glucoside (CG), or with a pH acidifier, citric acid (CA). Minispheres protected sCT from thermal degradation and the released sCT retained high bioactivity, as determined by cyclic AMP generation in T47D cells. Pre-minisphere emulsions of PEs combined with sCT increased absolute bioavailability (F) compared to native sCT following rat intra-jejunal (i.j.) and intra-colonic (i.c.) loop instillations, an effect that was more pronounced in colon. Minispheres corresponding to ~2000I.U. (~390µg) sCT/kg were instilled by i.j. or i.c. instillations and hypocalcaemia resulted from all prototypes. The absolute F (i.j.) of sCT was 11.0, 4.8, and 1.4% for minispheres containing NaTDC (10µmol/kg), CG (12µmol/kg) or CA (32µmol/kg) respectively. For i.c. instillations, the largest absolute F (22% in each case) was achieved for minispheres containing either C10 (284µmol/kg) or CG (12µmol/kg), whilst the absolute F was 8.2% for minispheres loaded with CA (32µmol/kg). In terms of relative F, the best data were obtained for minispheres containing NaTDC (i.j.), a 4-fold increase over sCT solution, and also for either C10 or CG (i.c.), where there was a 3-fold increase over sCT solution. Histology of instilled intestinal loops indicated that neither the minispheres nor components thereof caused major perturbation. In conclusion, selected SmPill® minisphere formulations may have the potential to be used as oral peptide delivery systems when delivered to jejunum or colon.

Targeted delivery of the hydroxylase inhibitor DMOG provides enhanced efficacy with reduced systemic exposure in a murine model of colitis.

Targeting hypoxia-sensitive pathways has recently been proposed as a new therapeutic approach to the treatment of intestinal inflammation. HIF-hydroxylases are enzymes which confer hypoxic-sensitivity upon the hypoxia-inducible factor (HIF), a major regulator of the adaptive response to hypoxia. Previous studies have shown that systemic (intraperitoneal) administration of hydroxylase inhibitors such as dimethyloxalylglycine (DMOG) is profoundly protective in multiple models of colitis, however the therapeutic potential of this approach is limited due to potential side-effects associated with systemic drug exposure and the fact that orally delivered DMOG is ineffective (likely due to drug inactivation by gastric acid). In order to overcome these issues, we formulated DMOG in a liquid emulsion drug delivery system which, when coated with specific polymer coatings, permits oral delivery of a reduced dose which is released locally throughout the colon. This colon-targeted DMOG formulation demonstrated increased relative colonic bioactivity with reduced systemic exposure and provided a similar degree of protection to systemic (intraperitoneal) administration at a 40-fold lower dose in DSS-induced colitis. In summary, targeted delivery of DMOG to the colon provides local protection resulting in enhanced efficacy with reduced systemic exposure in the treatment of colitis. This novel approach to targeting hydroxylase inhibitors to specific diseased regions of the GI tract may improve it's potential as a new therapeutic in inflammatory bowel diseases such as ulcerative colitis.

In-vitro characterisation of a novel celecoxib microbead formulation for the treatment and prevention of colorectal cancer.

OBJECTIVES: Colorectal cancer (CRC) is a life-threatening disease that can develop as a consequence of a sustained chronic inflammatory pathology of the colon. Although not devoid of side effects, the anti-inflammatory drug celecoxib (CLX) has been shown to exert protective effects in CRC therapy. The purpose of this study was to develop and characterise a novel CLX microbead formulation suitable for use in the treatment and prevention of CRC, which has the potential to minimise the side effects associated with CLX. METHODS: The study involved the assessment of the effectiveness of CLX formulations in an in-vitro cell model (HT29 cells) and a comparison of these effects to that of the marketed CLX product, Celebrex. Liquid CLX formulations were developed as precursors to microbead formulations. The effect of liquid CLX formulations on HT29 cell viability (MTT and flow cytometry apoptotic assays) and motility (scratch wound assay) were assessed and compared with the effect of Celebrex. A correlation between the in-vitro dissolution performance of the formulations and the effect in the cell model was also explored. Liquid CLX formulations were translated into an optimised CLX microbead formulation, and a colonic targeted sustained release coat (Surelease) was applied to the beads with the aim of producing a formulation for a future in-vivo study to compare the effect of the coated CLX microbeads versus Celebrex in the attenuation of CRC tumours and inflammation in a CRC murine model. The production of CLX microbeads was scaled-up using vibrating-jet encapsulation technology to allow for the development of an optimised dissolution profile to enable colonic release. KEY FINDINGS: In-vitro cell viability and motility were shown to be significantly reduced after treatment with CLX liquid formulations relative to the control, whereas the results for treatment with Celebrex were comparable with the control. Dissolution experiments and correlation analysis demonstrated that the formulations that showed a greater extent of drug release had reduced cell viability and motility. The CLX liquid formulations were translated into colon-targeted CLX microbeads suitable for use in a future in-vivo mouse study. CONCLUSIONS: These results represent a significant step forward in the chemopreventative treatment of CRC using CLX, as the microbead formulation developed suggests the possibility of presenting CLX in a format that has the potential to minimise gastrointestinal and cardiovascular side effects.

Microbeads: a novel multiparticulate drug delivery technology for increasing the solubility and dissolution of celecoxib.

The purpose of this study was to develop a novel multipaticulate drug delivery technology suitable for the delivery of pre-solubilized celecoxib to the gastrointestinal tract and more specifically to the colon. The solubility of celecoxib in a range of oils, surfactants and co-solvents was evaluated. Celecoxib was solubilized in mixtures of these vehicles to produce liquid formulations. The in vitro dissolution of these liquid formulations was assessed and the data obtained was used to design microbead formulations containing celecoxib dissolved within an emulsion/micellar solution core. Microbead formulations were optimized to increase drug loading, avoid precipitation and to achieve good in vitro dissolution performance. An optimized formulation with a celecoxib loading of 6% w/w was produced and yielded an in vitro dissolution result of 80% over 6 h. The structure of these microbead formulations was characterized using light microscopy to reveal a correlation between droplet size and dissolution performance.

Investigation of coco-glucoside as a novel intestinal permeation enhancer in rat models.

Due to instability in the GI tract and low intestinal permeability, peptides invariably have oral bioavailabilities below 1% and this has prevented the development of oral formulations. A mild plant-derived naturalalkyl polyglycoside (APG), coco-glucoside (CG), was studied for its capacity to enable rat intestinal permeation of the paracellular sugar marker, fluorescein isothiocyanate-dextran 4000 (FD4), across isolated rat jejunal and colonic mucosae mounted in Ussing chambers, as well as the polypeptide, salmon calcitonin (sCT) following intra-intestinal instillations in rats. 0.1% (w/v) CG enabled a 2.9-fold increase in the apparent permeability coefficient (Papp) of FD4 over the basal Papp across colonic mucosae, but it was without effect in jejunal mucosae. In situ intestinal instillations revealed that although sCT was absorbed across rat colonic loops to a greater extent than jejunal, CG still improved sCT absolute bioavailability(F) from both segments. Histopathology of rat intestinal mucosae following exposure to CG indicated only minor perturbation with adequate maintenance of secretory function. High content analysis(HCA) on Caco-2 showed that acute and chronic exposure to a range of concentrations of CG did not cause sub-lethal damage at concentrations at which it was effective as an enhancer. Overall, CG increased bioavailability of sCT across rat jejunal and colonic loops without indication of tissue damage. Thus, CG has potential as a safe and effective intestinal enhancer for oral delivery of proteins and peptides.

Type I cadherins are required for differentiation and coordinated rotation in Xenopus laevis somitogenesis.

In Xenopus laevis somitogenesis, somitic blocks undergo coordinated movements resulting in their detachment from the rest of the mesodermal ridge, followed by a 90 degrees rotation of the entire metamere. Here we investigated the function of type I cadherins in somitogenesis. Type I cadherins are Ca(2+)-dependent cell-cell adhesion molecules concentrated in the adherens junctions and highly expressed in the somitic tissue. We analyzed their role in somitogenesis by overexpressing either the intracellular (deltaE) and the extracellular (C-trunc) dominant-negative forms of cadherin. The resulting phenotype was a downward bend of the anterior-posterior axis in tadpole stage embryos. 12/101 antigen and X-Myo-D expression were altered. Microscopy revealed disorganization of the myotomes. Conversely, segmentation was conserved at the microscopic and molecular levels.